Mutations in presenilin (PS) genes cause early onset familial Alzheimer's disease (FAD) by increasing production of the amyloidogenic form of amyloid beta peptides ending at residue 42 (Abeta42). To identify a PS subdomain responsible for overproduction of Abeta42, we analyzed neuro2a cell lines expressing modified forms of PS2 that harbor an N141I FAD mutation. Deletion or addition of amino acids at the C terminus and Ile448 substitution in PS2 with the N141I FAD mutation abrogated the increase in Abeta42 secretion, and Abeta42 overproduction was dependent on the stabilization and endoproteolysis of PS2. The same C-terminal modifications in PS1 produced similar effects. Hence, we suggest that the C terminus of PS plays a crucial role in the overproduction of Abeta42 through stabilization of endoproteolytic PS derivatives and that these derivatives may be the pathologically active species of PS that cause FAD.

Presenilin (PS) genes linked to early-onset familialAlzheimer's disease encode polytopic membrane proteins that are presumed to constitute the catalytic subunit of ␥-secretase, forming a high molecular weight complex with other proteins. During our attempts to identify binding partners of PS2, we cloned CALP (calsenilin-like protein)/KChIP4, a novel member of calsenilin/ KChIP protein family that interacts with the C-terminal region of PS. Upon co-expression in cultured cells, CALP was directly bound to and co-localized with PS2 in endoplasmic reticulum. Alzheimer's disease (AD)1 is a progressive dementing neurodegenerative disorder characterized by a massive deposition of ␤-amyloid and tau-rich neurofibrillary lesions in the brains (reviewed in Ref. 1 and references therein). A subset of AD is inherited as an autosomal dominant trait, and mutations in three different genes have thus far been linked to early-onset autosomal dominant forms of familial AD (FAD). Among these, presenilin 1 (PS1) and PS2 account for the majority of the early onset FAD (1). PS1 and PS2 genes encode polytopic integral membrane proteins that are predominantly localized in intracellular membranes and span the membrane six to eight times.PS proteins undergo endoproteolysis to give rise to N-and C-terminal fragments, which are the preponderant forms of endogenous PS in vivo (2). These fragments form a heterodimer and are incorporated into high molecular weight (HMW) protein complexes (2-5) that are highly stabilized (t1 ⁄2 ϭ ϳ20 h; Ref.6), whereas holoproteins of PS are rapidly degraded (t1 ⁄2 ϭ ϳ2 h) (6, 7). The steady-state levels of PS fragments seem to be tightly regulated by competition for shared, but limiting, cellular factors, because overexpression of PS in transfected cells does not increase the overall level of PS fragments and replaces endogenous PS (8).PS plays an important role in the generation of amyloid ␤ peptides (A␤) by facilitating intramembranous ␥-cleavage of ␤-amyloid protein precursor (␤APP), as evidenced by the lack of A␤ production and accumulation of ␤APP C-terminal stubs in cells established from PS-null mice (9 -11). In contrast, FADlinked mutations in PS increase the production of highly fibrillogenic A␤42 (12-15), which is the initial and predominantly deposited A␤ species in AD brains (16, 17) and normally consists of only ϳ10% of total secreted A␤ (18). Moreover, genetic studies in invertebrates and PS-null mice suggested that ␥-cleavage-like proteolytic cleavage at site 3 to release Notch intracellular domain (NICD), which is the prerequisite for Notch signaling (reviewed in Ref. 19), also is facilitated by PS. Furthermore, recent findings that the two intramembranous aspartates within the 6th and 7th transmembrane (TM) domains of PS are required for ␥-secretase activities (20) and that transition state analogue ␥-secretase inhibitors specifically label PS fragments (21-24) strongly support the notion that the PS-containing macroprotein complex catalyzes ␥-cleavage and that PS may represent the catalytic ...

Mutations in presenilin (PS) genes cause early-onset familial Alzheimer's disease by increasing production of the amyloidogenic form of amyloid ␤ peptides ending at residue 42 (A␤42). PS is an evolutionarily conserved multipass transmembrane protein, and all known PS proteins contain a proline-alanine-leucine-proline (PALP) motif starting at proline (P) 414 (amino acid numbering based on human PS2) at the C terminus. Furthermore, missense mutations that replace the first proline of PALP with leucine (P414L) lead to a loss-of-function of PS in Drosophila melanogaster and Caenorhabditis elegans. To elucidate the roles of the PALP motif in PS structure and function, we analyzed neuro2a as well as PS1/2 null fibroblast cell lines transfected with human PS harboring mutations at the PALP motif. P414L mutation in PS2 (and its equivalent in PS1) abrogated stabilization, high molecular weight complex formation, and entry to Golgi/transGolgi network of PS proteins, resulting in failure of A␤42 overproduction on familial Alzheimer's disease mutant basis as well as of site-3 cleavage of Notch. These data suggest that the first proline of the PALP motif plays a crucial role in the stabilization and formation of the high molecular weight complex of PS, the latter being the active form with intramembrane proteolytic activities. Alzheimer's disease (AD)1 is a progressive dementing neurodegenerative disorder in the elderly characterized pathologically by the presence of senile plaques and neurofibrillary changes in the brains of affected individuals (reviewed in Ref. 1, and references therein). Senile plaques are composed of amyloid ␤ peptides (A␤) comprising ϳ40 amino acids that are proteolytically produced from ␤-amyloid precursor protein (␤APP). ␤APP is initially cleaved by ␤-secretase to generate a 99-residue C-terminal fragment (C99) that then is cleaved by ␥-secretase to generate A␤. A subset of AD is inherited as an autosomal dominant trait (familial AD: FAD). Genetic mutations in ␤APP genes that cosegregate with the clinical manifestations of FAD increase production of the amyloidogenic A␤42 species ending at Ala 42 (2); A␤42, which normally comprises only ϳ10% of total secreted A␤, aggregates much faster than the predominant A␤40 species (3), and A␤42 is the initially and predominantly deposited A␤ species in AD brains (4, 5). These data implicated a seminal role of A␤42 in the pathogenesis of AD.Mutations in presenilin (PS) 1 and PS2 genes are linked to the majority of early onset FAD. FAD-linked PS mutations affect ␥-cleavage of ␤APP leading to an increased production of A␤42 (1). In contrast, ablation of PS1 and PS2 genes in mice completely inhibited production of both A␤40 and A␤42, accompanied by accumulation of the ␤APP C-terminal stubs (i.e. C99 and C83) that are the direct substrates for ␥-secretase (6 -8). Furthermore, studies in Caenorhabditis elegans and Drosophila melanogaster, as well as in knockout mice, suggested that PS facilitates Notch signaling by activating the ligand-induced intramembranous proteol...

The transmembrane glycoprotein nicastrin is a component of presenilin (PS) protein complex that is involved in Q Q-cleavage of L LAPP and site-3 cleavage of Notch. PS undergoes endoproteolysis, and the proteolytic fragments are incorporated into the high molecular weight protein complexes that are highly stabilized. Here we show that Endo H-resistant, N-glycosylated form of nicastrin (p150-NCT) is highly stabilized and selectively bound to PS fragments. Moreover, loss-offunction mutations of nicastrin inhibited formation of fully glycosylated p150-NCT as well as stabilization of nicastrin, suggesting that glycosylation and stabilization of nicastrin polypeptides are tightly correlated with its function. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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